Measurement of Peptide‐MHC Interactions in Solution Using the Spin Column Filtration Assay

Søren Buus1, Sanne Lise Lauemøller1, Anette Stryhn1, Lars Østergaard Pedersen1

1 Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.4
DOI:  10.1002/0471142735.im1804s31
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes how peptide‐MHC complexes can be generated in vitro using affinity‐purified MHC and synthetic peptide. The unit first describes how the interaction between peptide and MHC interaction can be measured in an accurate, quantitative biochemical assay. This procedure has been optimized for efficient separation of free peptide and MHC‐bound peptide through a novel principle, termed “gradient centrifugation.” The first two support protocols describe how to set up a biochemical fluid‐phase binding reaction between peptide and MHC class I and class II, respectively. Also, an alternative procedure for setting up a biochemical fluid phase binding reaction between β2m and MHC class I is included. Finally a more versatile inhibition assay is described. The assay is simple and robust, and has several advantages compared to the classical gel‐filtration assay, including increased sensitivity and throughput. It also demands fewer resources both in terms of unique reagents and labor, and it generates less hazardous waste. Thus, the spin column gel‐filtration assay is ideal for routine work.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Peptide‐MHC Binding Determined by Gradient Centrifugation Spin Column Chromatography
  • Support Protocol 1: Peptide‐MHC Class I Binding Reactions
  • Support Protocol 2: Peptide‐MHC Class II Binding Reactions
  • Support Protocol 3: β2‐Microglobulin/MHC Class I Binding Reactions
  • Support Protocol 4: Peptide‐MHC Inhibition Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Peptide‐MHC Binding Determined by Gradient Centrifugation Spin Column Chromatography

  Materials
  • recipeRunning buffer (see recipe)
  • 75% slurry of Sephadex G‐25 (Pharmacia Biotech) in reciperunning buffer (∼150 g of gel material in 900 ml buffer)
  • Peptide‐MHC reaction mixtures (see protocol 2Support Protocols 1 to protocol 54) in microtiter plates
  • 100 × 1.8–mm glass capillary tubes
  • Cotton
  • 1‐ml automatic pipet tips (Greiner, catalog # 740290)
  • 4‐ml (70 × 11–mm) centrifuge tubes (Nunc, catalog # 3 40399)
  • Programmable centrifuge with swinging‐bucket rotor (eg. Sigma 4‐10P with rotor 11140)
  • γ scintillation counter

Support Protocol 1: Peptide‐MHC Class I Binding Reactions

  Materials
  • Purified MHC class I (unit 18.3)
  • 1 mg/ml purified human β2‐microglobulin (e.g., Sigma)
  • Phosphate‐buffered saline (PBS; appendix 2A2)
  • recipe7.5× protease inhibitor/detergent cocktail (see recipe; prepare fresh)
  • 5000 cpm/µl 125I‐labeled indicator peptide (unit 18.3) in PBS (freshly diluted)
  • 96‐well microtiter plates with V‐shaped wells
  • Plate sealers (ICN Flow)
  • 18°C incubator

Support Protocol 2: Peptide‐MHC Class II Binding Reactions

  Materials
  • Purified MHC class II (unit 18.3)
  • Citrate/phosphate buffer, pH 5.0/83 mM citric acid/ 133 mM Na 2HPO 4
  • Phosphate‐buffered saline (PBS; appendix 2A2)
  • recipe7.5× protease inhibitor/detergent cocktail (see recipe; prepare fresh)
  • 5000 cpm/µl 125I‐labeled indicator peptide (unit 18.3) in PBS (recently labeled and freshly diluted)
  • 96‐well microtiter plates with V‐shaped wells
  • Plate sealers (ICN Flow)
  • 18°C incubator

Support Protocol 3: β2‐Microglobulin/MHC Class I Binding Reactions

  Materials
  • Purified MHC class I (unit 18.3)
  • Phosphate‐buffered saline (PBS; appendix 2A2)
  • recipe7.5× protease inhibitor/detergent cocktail (see recipe; prepare fresh)
  • 5000 cpm/µl 125I‐labeled β2‐microglobulin (very recently and mildly labeled, freshly diluted; see recipe)
  • 96‐well microtiter plates with V‐shaped wells
  • Plate sealers (ICN Flow)
  • 18°C incubator

Support Protocol 4: Peptide‐MHC Inhibition Assay

  • Inhibitory preparation of interest
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Björkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., and Wiley, D.C. 1987a. Structure of the human class I histocompatibility antigen, HLA‐A2. Nature 329:506‐512.
   Björkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., and Wiley, D.C. 1987b. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512‐518.
   Brown, J.H., Jardetzky, T.S., Gorga, J.C., Stern, L.J., Urban, R.G., Strominger, J.L., and Wiley, D.W. 1993. Three‐dimensional structure of the human class II histocompatibility antigen HLA‐DR1. Nature 364:33‐39.
   Buus, S., Sette, A., Colon, S.M., Jenis, D.M., and Grey, H.M. 1986. Isolation and characterization of antigen‐Ia complexes involved in T cell recognition. Cell 47:1071‐1077.
   Buus, S., Sette, A., Colon, S.M., Miles, C., and Grey, H.M. 1987. The relation between major histocompatibility complex restriction and the capacity of Ia to bind immunogenic peptides. Science 235:1353‐1358.
   Buus, S., Stryhn, A., Winther, K., Kirkby, N., and Pedersen, L.Ø. 1995. Receptor‐ligand interactions measured by an improved spun column chromatography technique: A high efficiency and high throughput size separation method. Biochim. Biophys. Acta 1243:453‐460.
   Falk, K., Rötzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.‐G. 1991. Allele‐specific motifs revealed by sequencing of self‐peptides eluted from MHC molecules. Nature 351:290‐296.
   Garrett, T.P.J., Saper, M.A., Björkman, P.J., Strominger, J.L., and Wiley, D.C. 1989. Specifity pockets for the side chains of peptide antigens in HLA‐Aw68. Nature 342:692‐696.
   Germain, R.N. and Margulies, D.H. 1993. The biochemistry and cell biology of antigen processing and presentation. Annu. Rev. Immunol. 11:403‐450.
   Madden, D.R. 1995. The three‐dimensional structure of peptide‐MHC complexes. Annu. Rev. Immunol. 13:587‐622.
   Rupert, J., Sidney, J., Celis, E., Kubo, R.T., and Sette, A. 1993. Prominent role of secondary anchor residues in peptide binding to HLA‐A2.1 molecules. Cell 74:929‐937.
   Schaeffer, E.B., Sette, A., Johnson, D.L., Beckoff, M.C., Smith, J.A., Grey, H.M., and Buus, S. 1989. Relative contribution of “determinant selection” and “holes in the T cell repertoire” to T‐cell‐responses. Proc. Natl. Acad. Sci. U.S.A. 86:4649‐4653.
   Sette, A., Buus, S., Colon, S.M., Smith, J.A., Miles, C., and Grey, H.M. 1987. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells. Nature 328:395‐399.
   Stryhn, A., Pedersen, L.Ø., Romme, T., Holm, C.B., Holm, A., and Buus, S. 1996. Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent sub‐specificities: Quantitation by peptide libraries and improved prediction of binding. Eur. J. Immunol. 26:1911‐1918.
   Yewdell, J.W. and Bennick, J.R. 1995. Cell biology of antigen processing and presentation to major histocompatibility complex class I molecule‐restricted T lymphocytes. Adv. Immunol. 52:1‐123.
Key References
   Buus et al., 1986. See above.
  Contains the first description of the simple gel filtration assay.
   Buus et al., 1995. See above.
  Contains the description of the gradient centrifugation spun column assay.
   Falk et al., 1991. See above.
  First description of the elegant pool sequencing of MHC class I bound peptides to describe their specificities.
   Rupert et al., 1993. See above.
  Contains an excellent example of the use of direct binding of a panel of unrelated peptides to describe MHC class I specificities.
   Stryhn et al., 1996. See above.
  Shows how direct binding of peptide libraries can be used to obtain an unbiased and complete description of MHC class I specificity.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library